Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA).To compare the effectiveness of liposomal tobramycin or polymyxin B against sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated.Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations - DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold.P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous  P. aeruginosa being the most common species isolated Chronic bronchial infections caused by opportunistic pathogens in the lower respiratory tract are a major cause of health decline in the CF population P. aeruginosaAntibiotics like aminoglycosides and polymyxins have been used for the treatment of acute or chronic exacerbations in response to multi-drug resistant (MDR) bacteria, particularly Gram-negative bacilli such as P. aeruginosain vitro, the harsh environment of sputum containing factors produced by host and the microbes reduce their potential interactions with the targeted pathogens Clinical studies have shown the success of antibiotics used in inhalation therapy, alone or in synergism, to combat multi-drug resistant Liposomes are biodegradable delivery vesicles made up of single or multiple phospholipids in the range of several nanometers to micrometers P. aeruginosa in sputum compared to the free antibiotic suggesting its potency in CF lung infections.Our data demonstrate that liposomes are stable in presence of sputum and inhibitory factors. This data is encouraging as it displays the ability of lipid vesicles to protect the antibiotics from inactivation. The study shows that free tobramycin and polymyxin B, incubated with negatively charged inhibitory factors, is greatly inhibited compared to liposome-entrapped forms at higher concentrations. Liposome-entrapped antibiotics display higher reduction in CFU of endogenous Escherichia coli (O111:B4) lipopolysaccharide (LPS), and Staphylococcus aureus lipoteichoic acid (LTA) were purchased from Sigma Chemicals Co . Monomeric G-actin was prepared from an acetone powder of rabbit skeletal muscle in a non-polymerizing buffer . G-actin was then polymerized to F-actin with the addition of 2 mM MgCl2 and 150 mM KCl and gently shaken for 1 h at room temperature. Depending on the experiment, DNA, LPS and LTA were dissolved in double distilled H2O or in cation-adjusted Mueller-Hinton (CAMH) broth. Synthetic 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), Cholesterol (Chol), and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) were obtained from Northern Lipids Inc . Polymyxin B , and tobramycin , were diluted in Phosphate Buffered Saline solution .Human placental DNA, G-actin, P. aeruginosa (ATCC 27853) were purchased from PML Microbiologicals . Clinical isolate strains PA-48912-1, PA-48912-2, and PA-48913 were kindly obtained from the Clinical Microbiology Laboratory of Memorial Hospital  and grown to form biofilm as described elsewhere 6 cfu/mL in CAMH broth before addition to 96-well plates.Reference strains cT = 23°C; DPPC cT = 41°C), for 2 h and unentrapped drug was washed off twice by ultracentrifugation at 62000 g. This step ensures that the unentrapped drug (in the supernatant) is separated from the liposomal pellet and is aspirated from the formulation. The liposomal suspensions were diluted at room temperature and size and polydispersity index was automatically determined with the use of a NICOMP 270/autodilute Submicron Particle Sizer according to manufacturer instructions . The content of antibiotic entrapped in liposomes (after disruption with 0.2% Triton X-100) was measured by an established method as described previously for tobramycin and polymyxin B Liposome- entrapped tobramycin or polymyxin B was prepared from a lipid mixture of either DMPC or DPPC and Chol (molar ratio of 2∶1), respectively, by dehydration-rehydration method as described previously with slight modifications P. aeruginosa , a combination of DNA, F-actin, LPS, and LTA at a concentration of 1000 mg/L, and intact or autoclaved sputum. In experiments involving sputum, pooled CF sputum was either kept intact and diluted 1∶10 (w/v), or autoclaved for 10 min before mixing with CAMH broth. After incubation, aliquots of the mixtures were removed and centrifuged. Antibiotic presence in the pellet was assayed by the microbiological assay as described above, and the amount of antibiotic released from the liposomes was expressed as a percentage of the total antibiotic concentration at 0 h.The stability of antibiotics in the formulations was examined according to Mugabe et al. P. aeruginosa (ATCC 27853) was grown on CAMH agar overnight at 37°C. Single colonies were diluted and suspended in CAMH broth alone or with 2-fold dilutions of LPS, LTA, DNA and F-actin (125 to 1000 mg/L); 2-fold dilutions of DNA and F-actin (125 to 1000 mg/L); and 10-fold dilutions of LPS and LTA (1 to 1000 mg/L). Equal volumes of 100 µL were added to a 96-well plate to a final concentration of 1×106 cfu/mL. To each well, 100 µL of the free or liposome-entrapped antibiotic  was added and the plates were incubated for 3 h at 37°C. The incubation period and concentration chosen were adequate to allow liposome or free antibiotic-bacteria interaction and eradication. After incubation, the suspensions were kept cool on ice and bacterial suspensions were diluted 10–10000 folds in PBS. Wells treated in the absence of polyanions were plated as is, i.e., without any dilutions. Aliquots (100 µL) of each dilution were plated on CAMH agar and incubated overnight at 37°C. The cfu/mL values were then determined for each of the three independent experiments.Antibacterial activity of the formulations was measured in the presence or absence of polyanions found in the CF lung. To determine the ability of liposomes to retain their antibiotic activity, the MBC of the antibiotic formulations were determined in an 18 h period by a standard microbroth dilution assay in CAMH broth alone or with a mixture of LPS, LTA, DNA and F-actin at a fixed concentration of 1000 mg/L. MBC assay was performed as mentioned above with addition of 2-fold dilutions of the free or liposomal antibiotic formulations added to the 96-well plates. The final volume in each well was 200 µL, and PBS or CAMH alone were used as positive (no antibiotic) and negative (no bacteria) controls, respectively. Following incubation for 18 h, aliquots (100 µL) were aspirated from each well and subcultured on CAMH agar plates overnight. MBC was defined as the lowest concentration of the antibiotic that resulted in less than 30 cfu live bacteria/Petri dish.P. aeruginosa were pooled and frozen at −80°C in aliquots. To measure the effects of the formulations on endogenous P. aeruginosa, aliquots of sputum samples were also stored at 4°C and used within 24 h of collection. At the time of the experiment, the sputum samples were diluted 1∶10 (w/v) in CAMH broth and mixed with the antibiotic formulations to achieve concentrations ranged 1 to 512 mg/L. The mixtures were then incubated for 18 h at 37°C and the cfu/mL of live bacteria was determined according to the aforementioned protocol. The dilution of the samples should have affected the viscoelastic properties of the sputum, and this dilution was only done for easier handling and measurement of the sputum samples.The sputum samples were collected by spontaneous expectoration from nine CF patients following informed consent and a protocol approved by the Research Ethics Committee . Patients' age, name, treatments and exacerbation records were kept confidential. Sputum samples colonized with moderate to heavy growth of t-test, and P-values were considered significant when (*) p<0.05, (**) p<0.01, (***) p<0.001.All results were expressed as mean±S.E.M. obtained from three trials. Comparisons between free and liposomal formulations were made by ANOVA one-way post The entrapment efficiency of tobramycin in liposomes composed of DMPC/Chol (35 mg∶ 10 mg) was 2.47±0.19 mg/ml with a mean size of 293.7±41.1 nm, (polydispersity index of 0.70±0.12). Liposomes containing polymyxin B in DPPC/Chol (38 mg∶ 10 mg) had an entrapment efficiency of 0.4±0.02 mg/ml, with a mean size of 445.1±49.3 nm (polydispersity index of 0.91±0.06).Liposomal stability and antibiotic leakage in different environments including CF sputum at 3 or 18 h post-exposure are shown in To determine the inhibitory effects of DNA, F-actin, LPS, or LTA on the activity of antibiotics, different concentrations of these inhibitory factors were co-incubated with the antibiotic formulations during a 3 h pre-incubation first. The free and liposomal tobramycin at 2 mg/L killed all ATCC 27853 strain within 3 h, while the liposomal and free polymyxin B eradicated bacteria at 1 mg/L and 2 mg/L, respectively. The bactericidal activity for both groups of antibiotics in the presence of DNA, F-actin, LPS, and LTA at the concentrations of 125 to 1000 mg/L is shown in Separately, the inhibition of activity by DNA and F-actin, LPS and LTA were assessed. When DNA and F-actin were co-incubated with the tobramycin formulations , free toSince negatively charged polyanions hindered bactericidal activity in a short period of time (3 h exposure), their effect on the MBCs in an 18 h period were also investigated . MBC levTo test the efficacy of entrapped versus free antibiotics in the CF sputa, pooled sputum was diluted and incubated with increasing concentrations of tobramycin and polymyxin B for 18 h. As shown in in vitro. The stability will ensure a continuous presence of the antibiotic at the site of infection, and improves antibiotic bioavailability and biodistribution in vivoPolycationic antibacterial agents, like aminoglycosides and polymyxins, require self-promoted uptake pathways for entry and eradication of Gram-negative bacteria et al.et al.Polyanions like DNA and F-actin have strong affinity for their multivalent counterions and tend to aggregate (form bundles) in the presence of cationic antibiotics which block their bioactivity in vitroet al.With regards to polymyxin B, reports from studies have shown G-actin polymerization in the presence of polymyxin B et al.The binding of free bacterial surface components (e.g. LPS and LTA) to polycationic antibiotics like polymyxin B may be beneficial to the host in terms of suppressing inflammation however it will compromise the antibacterial effect of the antibiotic. Tobramycin and polymyxin B tend to interact with the bacterial lipid membranes as indicated by the results of this study where the bioactivity of both antibiotics was reduced when co-incubated with LPS/LTA. However, the bioactivity of the antibiotics within the liposomes fared better  althoughIf the lipid bilayers of liposomes can decrease antibiotic interactions with the polyanionic components found in CF lungs and reduce bacterial growth within a 3 h period much more strongly than free antibiotics, its long term advantage and presence in an 18 h period would be advantageous . Unfortuin vitro, we sought to compare the bactericidal activity of these formulations against endogenous P. aeruginosa in CF sputa to that of the free drug. As shown in et al.P. aeruginosa infection model. In our study, due to issues of confidentiality, we did not have access as to the clinical status of the patients or their pathology laboratory reports. Nevertheless, delivery of antibiotics via a liposomal system enhanced their antibacterial activity in sputum.In light of the higher bactericidal activities and lower inactivation of liposomal antibiotics in the presence of polyanionic components in vitro (18 h exposure). This decrease in efficacy appears to be the result of pro-longed interactions of the liposomes with the polyanionic factors found in sputum. As prophylactic and anti-inflammatory treatments are improving the lung function of CF patients, reduction in neutrophil inflammatory response and bacterial infections may reduce its lysis and the presence of charged macromolecules which tend to inactivate cationic antibiotics. As novel approaches proceed towards a cure for CF, research must also be directed on strategies that obstruct the presence and/or action of inhibitory factors associated with the disease. Future work in our laboratory will tend to focus on disruption of these negatively charged factors for increased liposomal penetration.Although liposome entrapment of antibiotics and their increased efficacy is not a novel finding, neutral liposome-entrapped antibiotics tended to be more bactericidal in sputum and in the presence of sputum components when compared to free antibiotics, but with reduced efficacies over a longer period of time


There is evidence of functional growth hormone (GH) deficiency, expressed by means of low insulin-like growth factor 1 (IGF-1) serum levels, in a subset of fibromyalgia patients. The efficacy of GH versus placebo has been previously suggested in this population. We investigated the efficacy and safety of low dose GH as an adjunct to standard therapy in the treatment of severe, prolonged and well-treated fibromyalgia patients with low IGF-1 levels.Twenty-four patients were enrolled in a randomized, open-label, best available care-controlled study. Patients were randomly assigned to receive either 0.0125 mg/kg/d of GH subcutaneously (titrated depending on IGF-1) added to standard therapy or standard therapy alone during one year. The number of tender points, the Fibromyalgia Impact Questionnaire (FIQ) and the EuroQol 5D (EQ-5D), including a Quality of Life visual analogic scale (EQ-VAS) were assessed at different time-points.vs. 8.25 ± 0.9). Similar improvements were observed in FIQ score (p < 0.05) and EQ-VAS scale (p < 0.001). There was a prompt response to GH administration, with most patients showing improvement within the first months in most of the outcomes. The concomitant administration of GH and standard therapy was well tolerated, and no patients discontinued the study due to adverse events.At the end of the study, the GH group showed a 60% reduction in the mean number of tender points (pairs) compared to the control group . Fibromyalgia is a common syndrome of non-articular origin, characterized basically by fatigue and widespread musculoskeletal pain, along with tiredness and sleep disturbances. Pain in at least 11 of 18 trigger point sites on digital palpation is the only condition required to fulfil the American College of Rheumatology (ACR) criteria for fibromyalgia ,3.The cause of fibromyalgia is not known. However, the identification of this syndrome as a single clinical entity has improved the knowledge of its pathophysiology. There is evidence that the syndrome is influenced by psychiatric conditions such as anxiety or depression . AdditioThere is some evidence of functional GH deficiency, expressed as low insulin-like growth factor 1 (IGF-1) serum levels, in a subset of fibromyalgia patients . There iCurrent consensus based on clinical trials establishes that the best available fibromyalgia treatment should include drugs, rehabilitation and psychological support. Therapies with proven efficacy in fibromyalgia includes tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRI), and opioid analgesics . RegardiWomen greater than 18 years old with severe fibromyalgia and abnormally low IGF-1 levels included in an exercise rehabilitation and psychological program and stable under standard intensive treatment for at least 6 months participated in this prospective, randomized, open-label, best available treatment-controlled clinical trial. All patients fulfilled the 1990 ACR diagnostic criteria [Exclusion criteria were as follows: Disabling physical or mental status; previous or current malignancies, either active or inactive; intracranial space occupying lesion; any relevant endocrine disorder including diabetes mellitus; history of another pituitary disorder; previous treatment with growth hormone; other systemic or joint inflammatory rheumatic conditions; and known to be hypersensitive to somatropin or any of the excipients. Pregnant women, nursing mothers, or women with childbearing potential not using adequate contraceptive methods were also excluded.This study was conducted in the Centro Médico Teknon between May 2004 and November 2005. The study was conducted in accordance with the Declaration of Helsinki and received the local institutional review board and Spanish Drug Agency (n°C03-0456) approvals. It has been registered (NCT00497562) at ClinicalTrials.gov. All patients gave written informed consent prior to their inclusion in the study.® 8 mg click easy, solution for injection). Patients were randomly assigned, according to a computer-generated randomisation table, to receive either 0.0125 mg/kg/d of r-hGH subcutaneously added to standard intensive therapy or standard intensive therapy alone during one year. Standard intensive therapy (administered under stable conditions for at least 6 months before inclusion) consisted of three oral administered drugs: amitriptyline 10–50 mg/d, fluoxetine (or other SSRI 10–40 mg/d) and tramadol 100–400 mg/d. These doses were individually adjusted according to the associated pathology in each case. Doses of r-hGH were adjusted after the first month according to IGF-1 plasma levels and/or the appearance of adverse events possibly related to GH.The study medication is a human growth hormone produced by recombinant DNA technology in a mammalian cell line  and a cranial magnetic resonance imaging (MRI) were performed at baseline visit at investigator's discretion when IGF-1 levels were < 150 ng/mL .Follow-up visits were scheduled at 1, 3, 6 and 12 months. At baseline and follow-up visits, investigators determined the number of tender points according to the ACR criteria for fibromyalgia and patients self-completed the FIQ, the EQ-5D, and a health-related Quality of Life Visual Analogic Scale (VAS). A blood sample was drawn at baseline and at follow-up visits to measure blood cell count, biochemical profile and other laboratory assays including GH, IGF-1, thyroid-stimulating hormone (TSH), thyroxine (free T4), triiodothyronine (free T3), Estradiol (E2), progesterone (P4), urinary free cortisol, insulinemia, apolipoprotein B, C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, aldolase and creatinkinase. Adverse events, concomitant medication and treatment compliance were recorded at each follow-up visit. A flow chart of the study procedures is shown in Table The primary efficacy endpoint was the reduction in the number of tender points at 12 months with respect to baseline. Fibromyalgia trigger points were assessed by an observer blind to the treatment group using the protocol described by Wolfe et al. employing 18  standardized sites and according to ACR criteria  and the ® 2000, DPC, Los Angeles, CA). The interassay coefficient of variation of GH and IGF-1 assays was 4.52% and 3.04%, respectively. The analytical sensitivity of GH and IGF-1 assays was 0.05 ng/mL and <25 ng/mL, respectively. Serum cortisol levels were also determined by chemiluminiscence using the Liason Analizer .Serum GH, IGF-1, TSH, free T4, free T3, E2, P4 and insuline levels were determined by automated chemiluminescent immunoassay system using a commercially available kit  and the investigator discretion.This was a pilot study with an exploratory goal. Thus, a sample size was estimated to obtain preliminary results that would allow evaluation for further possible investigations. Quantitative endpoints are presented as mean and standard deviation (SD) or median and range. The 95% confidence interval was used to indicate the precision of an estimate. The homogeneity of variances was analysed by a Levene's test and the within-group comparisons employed t test and the non-parametric Mann-Whitney Wilcoxon test when necessary. Categorical data are presented as absolute numbers and percentages. A Chi-Square analysis or Fisher exact-test were used to compare these variables when applicable.Regarding the main variable, the differences between groups were analyzed by analysis of covariance (ANCOVA), with basal number of tender points as a covariate. The time course within-group comparisons were analysed by repeated measurements analysis of variance (ANOVA). All p values were based on a two-tailed distribution, and 5% level of significance was considered. The statistical analysis was based on Intention to Treat. The SPSS statistical software package (version 12.0) was used for statistical analysis.The study population included 24 women with a mean age of 48.5 years, randomly assigned to r-hGH (n = 12) or control (n = 12) groups. All patients fulfilled the ACR criteria for fibromyalgia, the cut-off levels for severity (16/18 positive trigger points and FIQ>75) and duration of symptoms (>1 year), IGF-1 levels>250 ug/dL  and completed the study. GH levels were normal and comparable in both groups. Baseline data are shown in Table With regard to the primary efficacy outcome, the study showed a reduction in the number of tender points (pairs) in the r-hGH group compared to the control group, at 12-month evaluation (p = 0.0001)   in comparison to control group in both pain and fatigue FIQ subscales. Thus, for the FIQ-pain subscale, the mean (SD) was 8.67 (0.98) and 8.5 (1.09) at baseline, and 5.17 (2.44) and 7.75 (1.36) at 12-month evaluation in the r-hGH and control groups, respectively. A similar pattern of results was observed for the FIQ-fatigue subscale, with a mean (SD) of 8.75 (1.6) in both groups at baseline, and 4.92 (1.8) and 7.75 (1.2) at 12-month evaluation in r-hGH and control groups, respectively.There were also significant differences between treatment groups in terms of Quality of Life (EQ-5D). Thus, there was a significant improvement in the mean VAS score for the r-hGH group compared to control group at 1, 3, 6 and 12 months (p < 0.05), with a within-group significant improvement from month 1 until the end of the study compared to baseline (p = 0.001) Figure . With reNo withdrawals due to lack of efficacy were reported in the study.st month (p = 0.028). No further correlation was observed with IGF-1 or any other biochemical marker.A statistically significant correlation between the increase of IGF-1 serum levels and clinical improvement was observed at the 1In terms of tolerability, no important changes in any of the clinical or laboratory parameters were observed in any of the treatment groups. No withdrawals due to adverse events were reported in the study in any treatment group. Despite the high r-hGH dosage , only 10 adverse events were reported in 4 patients, all of them in the r-hGH group . No patient discontinued the study due to adverse events. All reported adverse events were considered to be possibly related to the study treatment. All events were of mild intensity and no serious adverse events were reported during the study.Fibromyalgia is a common syndrome of widespread musculoskeletal pain. Although its etiology is not known, there is evidence that fibromyalgia is influenced by several factors, including alterations of autonomic nervous system activity and HPA axis . Growth The standard pharmacological therapy for this syndrome includes the combination of tricyclic antidepressants, SSRI and opioid analgesics . CurrentThe present study is a prospective, randomized, open-label, best available treatment-controlled clinical trial. Even though no placebo group was included, the different overall response observed between treatment groups, as well as the intra-group differences in the r-hGH group as compared with baseline, supports the internal validity of this assay. The endpoints used in this study are well-validated outcome measures in the evaluation of fibromyalgia. The manual trigger point examination (in this case by an observer blinded to the treatment) has been historically considered a key feature in the definition of fibromyalgia Regarding the quality of life self-assessment, the VAS evaluation showed a similar trend, with a 38.7% mean score improvement at 12 months in the r-hGH group versus control group and 64.9% versus baseline (p = 0.001 in both comparisons). The weighted score assessment of the EQ-5D questionnaire at the end of the study compared to baseline showed a mean change of 0.59 and 0.33 in r-hGH and control groups, respectively. This change from baseline achieved statistical significance in the r-hGH group (p = 0.017).It is important to mention the prompt response in the r-hGH group, evidenced and maintained since the first month assessment. In our study all 12 patients in the r-hGH group experienced global improvement and 1 patient even scored 100 in the VAS scale at the end of the study. This contrasts favourably with Bennett's previous study [st month follow-up vist, which was normalized at the 3rd month evaluation after reducing the dose by 0.02 mg/d. No patients discontinued the study due to r-hGH related adverse events. No control group dropouts were seen, possibly due to the intense standard treatment received .The concomitant administration of r-hGH and standard therapy was well tolerated. No important changes in any of the clinical or laboratory parameters were observed in the two groups. A total of 10 adverse events were reported in 4 patients, all of them in the r-hGH group. However, only one (8.3%) carpal tunnel syndrome was described in comparison with the 7 cases (31.8%) reported in the GH treatment group in Bennett's previous study  despite The present study suggests the advantage of adding a 0.0125 mg/Kg r-hGH daily dose to the standard therapy versus the standard therapy alone in a subset of severe fibromyalgia patients with low IGF-1 serum levels. Obviously, the results of this study should be viewed in light of the methodological limitations, such as the open assignment to the study drug, the lack of a placebo group and the small sample size. However, the consistency and magnitude of these results, obtained in this well-characterized sample and with long-term treatment period, added to the similar trend observed in the previous placebo-controlled study, are encouraging and warrant further validation in a multicenter, double-blind confirmatory study which is currently ongoing.The authors have received external funding from Serono-Iberia (now Merck España S.L) for the preparation of study data for submission. The authors declare that they have no other competing interest.GC and AN designed the study, made the overall coordination and prepared the data for presentation. Both GC and CR visited the patients in the endocrinological view. CR passed the EQ-5D questionnaire and trained the patients to the use of GH. GC made the blind randomization, up or down-titrate the GH dose according to IGF-1 or adverse effects . MAG performed the psychological assistance required in trial protocol in both groups. AN visited the patients in the reumatological view, assessed the tender points and administered the VAS and FIQ scale and subscales (blinded to the treatment groups) and also evaluated the fibromyalgia standard therapy dosage. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Real-Time quantitative PCR is an important tool in research and clinical settings. Here, we describe two new approaches that broaden the scope of real-time quantitative PCR; namely, run-internal mini standard curves (RIMS) and direct real-time relative quantitative PCR (drqPCR). RIMS are an efficient alternative to traditional standard curves and provide both run-specific and target-specific estimates of PCR parameters. The drqPCR enables direct estimation of target ratios without reference to conventional control samples.In this study, we compared RIMS-based drqPCR with classical quantifications based on external standard curves and the “comparative Ct method”. Specifically, we used a raw real-time PCR dataset as the basis for more than two-and-a-half million simulated quantifications with various user-defined conditions. Compared with classical approaches, we found that RIMS-based drqPCR provided superior precision and comparable accuracy.The obviation of referencing to control samples is attractive whenever unpaired samples are quantified. This may be in clinical and research settings; for instance, studies on chimerism, TREC quantifications, copy number variations etc. Also, lab-to-lab comparability can be greatly simplified. N0 is the amount of the target sequence before PCR, CqN is the amount of target after qC-rounds of PCR, and E is the efficiency of the PCR-amplification. Usually, E is assumed constant until the onset of PCR exhaustion. The designation “relative quantification” refers to the fact that the amount of the target sequence is estimated relative to that of another  can thus be estimated as:CqNs, qCs and Es must be accounted for. Indirect measures of CqNs are estimated by fluorescence sampling. Various technologies exist  has long been a favoured principle for relative quantifications of nucleic acid sequences. In essence, an undetectably low amount of a specific nucleic acid target sequence is expanded by PCR to a measurable level. Subsequently, the original amount of the target sequence is calculated from the parameters of the PCR. The basis of these calculations is the classical PCR equation:Despite the broad applicability of the technology, several methodological limitations have yet to be addressed. In this paper, we focus on two of these.Eq.I.2 cannot be solved trivially offhand. The problem lies in the Cq,AN/Cq,BN-term; in conventional settings, the value of this term is unknown. This may not be problematic as such, provided that the ratio of A and B in the interest-sample is of interest only relative to their ratio in a control-sample (cs):Cq,AN/Cq,BN-term, if both CqN and E are preserved for each target between the samples is and cs; that is: (CqN)A,is/(CqN)B,is = (CqN)A,cs/(CqN)B,cs and A,isE = A,csE, B,isE = B,csE):Eq. I.4 is generally unsuitable if samples are unpaired: the ratio of A and B in a sample can be of immediate interest, and any reference to a control sample can be inconvenient or even meaningless. In these situations, Eq. I.4 can still be useful if the control sample contains A and B in equal numbers. Nonetheless, two problems remain. First, such control samples are not necessarily available. Second, and more importantly, the sources of errors increases by the doubling of sample numbers to be determined with Eq. I.4 compared with Eq. I.2. Ultimately, increased error of the final ratio-estimate is very likely. It may therefore be attractive to actually use Eq. I.2 directly. If so, the inherent (CqN)A/(CqN)B-term must be accounted for. Of course, if (CqN)A and (CqN)B happen to be equal, the term disappears, but this situation is unlikely to occur by chance. Simply assuming equality  induces proportional errors of the isR-estimate. “Ensuring” equality by assigning the same fluorescence-threshold-value for A and B is unreliable. The proportionality factor between fluorescence and sequence-numbers (k) differs widely between targets. For example, the k-value of the fluorophore SYBRGreen depends on the length and sequence of the amplicon as well as other factors, such as salts and temperature CqN)A/(CqN)B-estimates, yet conventional approaches fall short in this respect.A general limitation is that E) from standard curves analysed in separate runs or assuming a value of 1. This introduces a run-to-run variability that inevitably contributes to the error of E. As such, E varies considerably between replicate runs . In this light, errors associated with run-to-run variability of PCR unknowns are highly undesirable.Another important limitation of conventional approaches concerns the use of low-capacity machinery. Conventionally, limited instrument capacity forces the investigator to estimate PCR unknowns  is superior to run-external estimation. Also, by modifying the composition of standard curve samples, we hypothesized that target ratio estimates can be attained directly without reference to control samples.Our objective was to deduce the optimal composition of standard curve samples to remedy the limitations of classical qPCR. In the process, we wanted to compare the precision and accuracy of our approach to classical approaches.Blunt-ended PCR products of parts of Human Endogenous retrovirus 1 (ERV1) and TUP-like enhancer of SPLIT 1 (TUPLE1) were produced from genomic DNA by conventional PCRs (Platinum Pfx DNA Polymerase (InVitrogen)). Primer sequences were obtained from Overhauser J et al The diluted fusion-PCR product was thoroughly mixed and stored in aliquots (−20°C). On three separate days, an aliquot was thawed and used for a 10-fold dilution series in eight steps. Weight data of the pipetted volumes were sampled while the dilution series were made. Each dilution step was performed three times into the same tube (to minimize impact of stochastic errors).Each dilution series was analyzed by real-time PCR with the primer pairs for ERV1 and TUPLE1. Each primer pair was used in separate runs. Reactions of 20 µl were set up in LightCycler capillaries: 10 µl 2× QuantiTect SYBR Green PCR mix (Qiagen), 0.5 µM primers and 8 µl template. PCRs were conducted on a LightCycler 1.0 Instrument (Roche) with the following settings: 15 min at 95°C, 45 amplification cycles . Each of the eight concentrations of the dilution series was analysed in four replicates. Six preliminary data sets containing 32 data points each were thus generated.qCs for the six preliminary data sets by the fit-points-approach, which gave more linear standard curves than the second derivative maximums-method . In practice, arithmetic baseline adjustment was used, noise bands were set by default, and the lowest (eighth) concentration that yielded qCs for all replicates was excluded (explained below). The remaining data points of the six separate data sets  were used in the “minimize error”-function of the LightCycler software (version 3.5) for threshold setting. The fit-point number was selected for each of the six data sets as the number providing the smallest error. Relative target concentrations (N0s) in the dilution-series-samples were determined from the loaded volumes unless otherwise stated.We estimated qC- and N0-data were used to generate standard curves. We excluded data obtained from the eighth dilution step to avoid introduction of heteroscedasticity, which would invalidate conventional linear regression analysis . The data of the eighth concentrations thus merely represented a safety feature and were not used for further analysis. Standard curves based on relative target concentrations were defined as:N0) as the outcome variable and qC as the predictor variable is opposite of the conventional approach and may seem awkward. However, as N0 of the-samples-to-be-quantified are to be estimated as regression estimates from qC the perception makes sense. More importantly, estimating errors of isR is simpler as detailed in later sections and Appendix S1 section 2. Thus, the formula for the regression equation is:α and β being intercept and slope, respectively. Data of the six 28-sample- standard curves generated from fusion-PCR products are presented in Data handling was done in Microsoft Excel 2007. Sampled qC and N0) of the six 28-sample standard curves were used to generate 8,694 different RIMS. Each RIMS was based on raw data from two different concentrations of the same 28-sample standard curve data. In other words, each concentration was perceived as a separate RIMS sample (see C. RIMS-concentration-pairs could be chosen in 7·(7−1)/2 = 21 different ways from each 28-sample standard curve data set and in 6·21 = 126 ways using all six data sets. For each of the seven concentrations of a 28-sample-SC, four qC-replicates were available. To simplify our simulation, we decided to use an equal number of qC-replicates for the two concentrations in the individual RIMS. Thus, for a given RIMS-concentration-pair, 4!/(1!·(4−1)!))2+4!/(2!·(4−2)!))2+4!/(3!·(4−3)!))2+4!/(4!·(4−4)!))2 = 69 different RIMS could be created, for a grand total of 8,694 ( = 126·69) RIMS using all six 28-sample-SC data sets. For each RIMS, the α (intercept) and β (slope) were estimated by conventional linear regression with qCs as the predictor variables and the relative concentrations as outcome variables.To examine the usability of RIMS and RIMS-based drqPCR, we simulated a large number of individual quantifications from actual real-time PCR data. First, we combined the raw data (mple see . The relqC-measurement of each target per quantification). The qC-measurements included in the applied RIMS were not quantified using the particular RIMS. Also, a RIMS-set, i.e. one of each target, is necessary for quantification. Therefore, (28−m)·(28−n) different unicate quantifications cound be constructed from a RIMS-set (m and n being the number of qC-measurements in the two RIMS). Because more than 18 million RIMS-sets could be constructed in this manner 2), we introduced the constraint that each RIMS-set of ERV1 and TURPLE1 must encompass qC-measurements of samples positioned similarly in the LightCycler carrousel  RIMS-sets were generated. The α and β of these sets were used with Eq. R.1 and Eq. R.2 for direct relative quantification of ERV1 and TUPLE1 in the remaining samples in the run that was not included in the RIMS. The total number of quantifications were 2,500,848 ( = (7·(7−1)/2)·(42·(28−1·2)2+(4·3/2)2·(28−2·2)2+42·(28−3·2)2+12·(28−4·2)2)·3). To facilitate evaluation of accuracy and precision, we normalized the individual ratio-estimate by the theoretical ratio. The theoretical ratio was inferred from the quantified samples relative concentration based on data from the construction of the dilution series. This parameter, is,NormR, ideally equals 1. Any systematic discrepancy reflects inaccuracy whereas stochastic variation around the mean estimate reflects precision.The constructed RIMS were used in simulations of 2,500,848 direct relative quantifications (i.e. quantifications without reference to a control sample). To simplify, unicate quantifications were applied . Samples of identical position of ERV1 and TUPLE1 for each serial dilution were used as control samples, and the remaining combinations of samples used as interest samples. This provided 61,236 (272·28·3) double-ratios to be estimated. These were normalized by the theoretical ratio as above. X-values were determined as above.Double-ratio-based quantification was used  normalized double-ratios were calculated.The external-SC-based quantifications were done as the 2t-distributions. Probability testing of variance similarity  and two-tailed p-values = 2·P(F≥obsF). Significance level was set to 0.001 to avoid importance of mass significance.Regression analysis was based on least-squares methods and isR directly (without reference to a control sample), the unknown (CqN)A/(CqN)B-term presents a challenging. A simple solution is to perform real-time PCR on a sample containing A and B in equal concentrations. The (CqN)A/(CqN)B-term can then be estimated from the qC-data by rearrangement of Eq. I.2, provided that E-estimates are available (e.g. from standard curves). More elegantly, (CqN)A and (CqN)B can be inferred from the intercept of As and Bs standard curves (α = LOG (CqN), cf. Eq. M.1). If the underlying relation between relative and absolute scales is similar for A and B, then a meaningful estimate of the (CqN)A/(CqN)B-term can be determined from the intercepts. Similar scales can be attained simply by constructing the standard curves from a common sample containing same concentrations of A and B. If such standard curves is used then it is unnecessary to estimate the actual (CqN)A/(CqN)B-term. Instead isR can be estimated as a difference of regression estimates (from Eq. M.2):isR) (see Appendix S1 section 2). A sample containing A and B in equal concentrations is sometimes available. If not, the sample can be constructed; for example, by cloning A and B into the same plasmid. Instead of this somewhat cumbersome cloning approach, we recommend joining the targeted PCR products in a fusion-PCR product containing A and B in equal stoichiometry. This fusion-PCR product can be expanded endlessly by PCR and allows standard curves of maximal dilution ranges. A fusion-PCR product of ERV1 and TUPLE1 was constructed as a proof of principle and used in the present study .To estimate Eq. R.1 hinges on use of valid estimates of slopes and intercepts. These parameters can vary significantly between targets but also between PCR runs of the same target (Tabel I). Therefore, target-specific and run-specific parameters are preferable. We hypothesized that internal standard curves based on fewer samples are preferable over larger, external standard curves. This hypothesis was confronted as follows:The accuracy of 0N)A,is) and LOG ((0N)B,is) in Eq. R.1. Hellemans et al point out the error of the slope in linear regression is reduced by expanding the range of the dilution and including more measurements points Eq. S2.1 in Appendix S1). A large number of measurement points are not desirable with RIMS. However, it is deductable that predictor variable extremes reduce the error more effectively than those close to the predictor variable mean. We therefore based our strategy solely on “extreme concentrations” and used only two samples, of relative concentration C, for our RIMSs. Our next step was to determine the importance of C-size and number of replicate analyses of the two RIMS-samples for the precision of α- and β-estimates. The total of 8,694 different RIMS was constructed from the raw data of six 28-sample standard curve data sets presented in Material and Methods). The precisions of the RIMS-based α- and β-estimates, compared with those of external standard curves, are presented in C. The benefit of increasing the RIMS-sample replicate number was less obvious.Initially, we sought an optimal sample composition strategy for RIMSs. The composition should minimize the errors of regression estimates of LOG  can provide isR-estimates if the control sample contains A and B in equal concentrations. Using Eq. R.1, LOG (isR) can be estimated as:αs and βs are constant between interest and control samples for each target.Next, we investigated the quantitative precision and accuracy of our two approaches when used in combination. In total, 2,500,848 unicate quantifications were determined from the raw data of the six 28-sample data sets. The quantitative precision is summarized in Eq. R.1 and Eq. R.2). Fewer different samples are required in the former approach. This confers fewer sources of errors to isR. However, erroneous β are expectedly more critical in the single-ratio approach, because β is multiplied by larger values (full qCs as opposed to ΔqCs in the double-ratio approach). In the double-ratio approach, errors in β-estimates become less critical when ΔqCs decreases. A potential drawback of double-ratio drqPCR is that more capacity is required. However, this may be circumvented by using one of the RIMS samples as control sample also. The control sample should be chosen as the RIMS sample providing the smallest ΔqCs for the individual target in the given quantification. Naturally, one RIMS sample may be closest to A while another is closest to B. Acknowledging, a “virtual control sample” can be constructed for the specific interest sample-to-be-quantified comprised of qC- and N0-data from the closest RIMS sample . Correction for the situation where (0N)A,cs≠(0N)B,cs is remedied using by the following equation:Some heterogeneity is evident in a comparison of single-ratio to double-ratio based drqPCR (C≥103). We expected that increasing the ΔqC of interest sample and virtual control sample would decrease the precision of double-ratio drqPCR. Data were therefore split according to X, which is a measure of this distance (Eq. M.3). Results are presented in Cs (103–104), the double-ratio approach was more precise when target concentrations of control and interest samples were close , we observed no remarkable differences between the approaches. The accuracy of both approaches was within ±8%.We compared the precision and accuracy of quantification by single- and double-ratio drqPCR in the 1,190,880 possible quantifications from the six 28-samplestandard curve data sets . Estimates determined for the specific run are obviously preferable to those determined in separate runs. Run-specific differences of CqN for the targets can be corrected by inclusion of calibrator samples. However, run-to-run differences in E are not corrected by use of calibrator samples and are critical to the use of external standard curves. It is evident from our data that run-to-run differences of E are to be anticipated .Separately, RIMS gives target-specific and run-specific estimates of the standard curve's slope and intercept  and 2.5% (TUPLE1). The CVs of CqN in the study of Rutledge and Cote were 19.0% and 14.7%. CqN-detection was based on constant fluorescence threshold. We based our threshold setting on an error-minimizing strategy for the individual standard curve. Correction of our CqN -data by fluorescence intensity at qC enables comparison. Threshold-corrected CqN CVs of our study were 12.4% and 26.3%  of the large- curves' Figure 1C (≥105) while preserving linearity. Second, very dilute samples should be avoided completely . Third, the chosen samples should be aliquoted and stored. Fourth, two or more replicate qC-estimations of each RIMS sample are preferable. Fifth, the type of template  chosen for RIMS-samples should permit appropriately sized Cs. Direct estimation of nucleic acid sequences ratio is attractive in many settings, yet difficult to obtain. The problem, associated with the (CqN)A/(CqN)B-term (cf. Eq. I.2), can be dealt with in several ways. The simplest is to ignore it altogether. This is the case if ordinary relative standard curves are used. Such an approach confers systematic errors of magnitudes defined by the inverse of the (CqN)A/(CqN)B-term. In our example, we would have attained between 1.1-fold and 2.6-fold errors for same-day quantifications and as much as 20-fold errors for quantifications across days A and (CqN)B. If equality is indeed ensured, the (CqN)A/(CqN)B-term can be safely ignored. A frequent perception in Real-Time PCR literature is that a similar detection threshold for different targets automatically ensures similar copy numbers at detection k) can differ between targets. The magnitude of the systematic error associated with the approach can be assessed as the inverse of the targets k-ratio. With this approach, our results would have been off by factor of 1.2 to 1.6 or 2.0 to 2.7 with or without target length corrections, respectively A/(CqN)B-term variability can provide results of reasonable precision. But the systematic error is never spotted unless samples of known ratio are quantified. A third and more qualified approach is to use classical absolute quantifications The following points should be considered when constructing the RIMS in practice. First, RIMS samples should be selected from the dilution series based on a preliminary standard curve to maximize CqN)A/(CqN)B. Ligated PCR products of the targets are the ideal theoretical choice. This generally applicable method has the potential for attaining the largest Cs and absolutely defining the targets' stoichiometry. For data handling, we suggest two different approaches: single-ratio or double-ratio-based drqPCR . In the double-ratio-based approach, a virtual control sample is constructed from the RIMS samples already used for β-estimations. Such “recycling” of RIMS-samples is not problematic, inasmuch as the samples are used to estimate two independent quantities. The known relative concentration between the RIMS-samples (e.g. AC = (N0)A,RIMS1/(N0)A,RIMS2) is used for β-estimations, whereas the relative concentration of the targets of the virtual control sample (e.g. (N0)A,RIMS1/(N0)B,RIMS1)) is used in corrections of the (CqN)A/(CqN)B-term. Steps to reduce the error of isR-estimation are of general importance. This is obvious in clinical settings but also applies to experimental comparison of groups. The larger the variance of a group mean, the more individuals, cell cultures or such, must be included to demonstrate a given significant difference between groups. With this in mind we allocated some efforts to choose the ideal raw data sampling approaches. qC-sampling by the FP-approach offered significantly better precision than the second derivatives maximum-approach, whereas estimations of relative target concentrations in standard curves based on weight as opposed to volumes was negligible (and insignificantly) better .Instead, we advocate approaches based on internal, relative standard curves  and use of regression estimates. This ensures both target-specific and run-specific corrections of the underlying variable .We evaluated drqPCR based on RIMS in quantifications of samples containing the quantified targets in known stoichiometry. This model permitted us to evaluate both quantitative precision and accuracy. Not surprisingly, we found that increasing icantly –3. HoweqN-value. Briefly, the calibrator sample contains the targets-to-be-quantified and is PCR-expanded both in runs of controls and interest samples. Subsequently, interest and control data are made comparable by dividing each with the calibrator data of their respective runs. In drqPCR, data of control and interest samples are always immediately comparable, provided that the same RIMS samples are used in runs. Avoidance of calibrators is attractive to minimize the sources of errors of isR. The importance of drqPCR can be stretched further. Another important effect is that results obtained by drqPCR are immediately comparable between different labs. Thus, the problem of lab-to-lab comparability is avoided completely.Use of drqPCR has other beneficial side-effects. The principle renders calibrator samples (or reference-control samples) superfluous. Calibrators are a necessity when interest and control samples are not in the same PCR-run isR directly in the sample, and RIMS can replace external standard curves.In summary, we suggest that RIMS and drqPCR be used separately or combined for relative quantifications of high precision and accuracyThe drqPCR allows determination of Appendix S1(0.12 MB DOC)Click here for additional data file.Figure S1qC spread and 0CqN residuals were calculated  and plotted against the sample number (1–8) of the dilution series. Levene's test for equality of qC-residuals' variances between groups where conducted. Inclusion of data of the eighth samples was associated with heteroscedasticity (p: 6·10−11) whereas exclusion conferred homoscedasticity (p: 0.66). NB: On this single occasion, we made two modifications to our qC-sampling approach. Firstly, noise bands were set manually for 3 out of 6 data sets. This was necessary to ensure that thresholds were not set in the lower non-logarithmic phases. Secondly, qCs of all eight concentrations of the dilution series were used in the minimizing error strategy.The association of replicate-(0.21 MB TIF)Click here for additional data file.Figure S2qC-sampling by SDM and FP  for different values of C . qC-sampling by FP prompted significantly (p<0.001) better quantitative precision for all values of C.Precision of RIMS based single-ratio drqPCR with (0.11 MB TIF)Click here for additional data file.Figure S3ΔΔCq-approach (black and dark grey bars) or from external standard curves (light grey and white bars) for variable sizes of X (defined in main text). qC-sampling was by SDM (black and light grey bars) or FP (dark grey and white bars). Quantifications based on qC-sampling by FP were more precise for both approaches of efficiency estimations for all X-values. The improvements were significant (p<0.001) except for external standard curves and X of 5 (p: 0.035) and 6 (p: 0.066) (indicated by asterisks).Precision of double-ratio-based qPCR by the 2(0.18 MB TIF)Click here for additional data file.Figure S4C based on pipetted weights (white and dark grey bars) or volumes (black and light grey). Weight as opposed to volume based C-estimation provided minute improvements of precision for all Cs regardless of qC-sampling approach . However, improvements were insignificant except where indicated by an asterisk (p: 0.0002). Analysis of RIMS samples in replicates of 1, 3, and 4 demonstrated similar findings.Quantitative precision of single-ratio drqPCR based on RIMS  with estimations of (0.13 MB TIF)Click here for additional data file.Algorithm S1Algorithm for practical use of RIMS based drqPCR.(1.26 MB XLS)Click here for additional data file.


Plasmodium falciparum, a member of a newly identified family of phosphoethanolamine methyltransferases (PMT) found solely in some protozoa, nematodes, frogs, and plants, is involved in the synthesis of the major membrane phospholipid, phosphatidylcholine. PMT enzymes catalyze a three-step S-adenosylmethionine-dependent methylation of the nitrogen atom of phosphoethanolamine to form phosphocholine. In P. falciparum, this activity is a limiting step in the pathway of synthesis of phosphatidylcholine from serine and plays an important role in the development, replication and survival of the parasite within human red blood cells.The phosphoethanolamine methyltransferase, PfPMT, of the human malaria parasite in vitro. Interestingly, PfPMT activity was not inhibited by the amodiaquine analog, chloroquine, or other aminoquinolines, amino alcohols, or histamine methyltransferase inhibitors. Using yeast as a surrogate system we found that unlike wild-type cells, yeast mutants that rely on PfPMT for survival were sensitive to amodiaquine, and their phosphatidylcholine biosynthesis was inhibited by this compound. Furthermore NMR titration studies to characterize the interaction between amoidaquine and PfPMT demonstrated a specific and concentration dependent binding of the compound to the enzyme.We have employed an enzyme-coupled methylation assay to screen for potential inhibitors of PfPMT. In addition to hexadecyltrimethylammonium, previously known to inhibit PfPMT, two compounds dodecyltrimethylammonium and amodiaquine were also found to inhibit PfPMT activity in vitro and in yeast, and the biophysical evidence for the specific interaction of the compound with the enzyme will set the stage for the development of analogs of this drug that specifically inhibit this enzyme and possibly other PMTs.The identification of amodiaquine as an inhibitor of PfPMT  Plasmodium. Of the five species infective to humans, Plasmodium falciparum is responsible for the highest level of mortality and morbidity [P. falciparum phospholipids are excellent targets for the development of lipid-based antimalarial therapies [Malaria is one of the most important parasitic diseases worldwide responsible for over 200 million clinical cases with an estimated 1 million deaths annually [herapies .P. falciparum demonstrated that the synthesis of phosphatidylcholine during the intraerythrocytic life cycle of the parasite occurs via two pathways, the serine-decarboxylase phosphoethanolemine methyltransferase (SDPM) pathway and the CDP-choline pathway [Studies in  pathway -4. The S pathway ,5,6, whiC. elegans expresses two PEAMT enzymes each containing only one methyltransferase domain located at either the N-terminus of the protein, in the case of Pmt1, or at the C-terminus of the protein in Pmt2 [Plasmodium PfPMT is only half the size of the plant and nematode proteins, possesses a single catalytic domain, and catalyzes all three methylation steps [The 266 amino acid PfPMT is a member of a new class of SAM-dependent methyltransferases that acts on P-EA . Homolog in Pmt2 ,11. Pmt1on steps . No crysP. falciparum, hexadecylphosphocholine was found to inhibit the growth of the parasite within human erythrocytes with 50% inhibitory concentrations in the low micromolar range [P. falciparum demonstrated that PfPMT plays an important role during the intraerythrocytic life cycle of the parasite [Confocal and immunoelectron microscopy studies have shown that PfPMT is expressed in the Golgi apparatus of the parasite . Biochemar range . Geneticparasite . Parasitparasite . These dHere we provide biochemical and biophysical data indicating that PfPMT activity is inhibited by amodiaquine (AQ). Using yeast as a surrogate system we show that AQ inhibits PfPMT activity and blocks PtdCho biosynthesis.S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), Phosphoethanolamine (P-Etn), Cholorquine (CQ), Amodiaquine (AQ), Hexadecyltrimethylammonium bromide (HDTA), Dodecyltrimethylammonium bromide (DDTA), 2,3-Dichloro- a- methylbenzylamine (DCMB), Chlorpromazine, and Diphenhydramine were purchased from Sigma. Miltefosine (HePC) was purchased from Cayman chemicals. SKF91488 and Tacrine were purchased from Tocris Bioscience. Recombinant SAH nucleosidase (SAHN) was purified from an E. coli strain generously given by K. Cornell , as previously described [P. falciparum PfPMT cDNA cloned in the expression vector pET-15b and expressed in E. coli BL21-CodonPlus strain was purified as described earlier [E. coli was grown until an A600 ~ 0.6, and PfPMT expression was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranosidase (IPTG). The cells were harvested 3 h after induction by centrifugation, and resuspended in 25 ml lysis buffer  containing a protease inhibitor cocktail . The cells were lysed by sonication using 10 s discontinuous cycles for three minutes on ice, and the cell debris and unbroken cells were removed by centrifugation at 10,000 rpm at 4°C for 30 min. The supernatant was directly applied to a 5 ml Ni-NTA (Qiagen) column pre-equilibrated in lysis buffer. The column was subsequently washed with 40 ml of buffer  and the protein was eluted from the column in a single 15 ml fraction of buffer . The protein was incubated at 4°C for 1 h in the presence of 2 mM Na2EDTA, followed by dialysis and concentration against HEPES (N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid) assay buffer  using Amicon Ultra centrifugal filter devices. The purity of the recombinant enzyme was examined by SDS-PAGE, and the protein concentration was measured by the method of Bradford using bovine serum albumin as a standard. earlier ,6. ExpreBacillus subtilis adenine deaminase (BsAda) was amplified from genomic DNA by PCR and ligated into a pET15b plasmid vector between XhoI and BamHI sites to yield an N-terminal 6His-epitope-tagged encoding construct. The resulting plasmid was then transformed into E. coli BL21-CodonPlus strain for enzyme expression. The purification protocol for BsAda is similar to PfPMT purification except that the 1 hr incubation step with 2 mM Na2EDTA was omitted.The gene encoding Assays were performed in 96-well UV-transparent plates  at 37°C. Manganese sulfate (MnSO4) was added to a final concentration of 1 mM . The ass2O, except for HDTA, which was prepared in DMSO. The final concentration of DMSO in the assays did not exceed 10%. Control experiments containing reaction components and inhibitors were performed in the absence of PfPMT to quantify the intrinsic absorbance of inhibitors at 265 nm. These inhibitors were added before the pre-incubation step. The absorbance at 265 nm for a PfPMT assay without inhibitor was assigned as 100% PfPMT activity. The absorbance values obtained for the various inhibitor concentrations were then converted into a PfPMT activity percentage and plotted against inhibitor concentration. Control experiments were performed to ensure that the inhibitors did not affect the coupling enzymes. To measure coupling enzyme sensitivity to inhibitors, 100 μM SAH was used in place of SAM and P-EA in a 200 μl reaction in the presence of the inhibitor at the highest concentration used in PfPMT assay. For Ki determination enzymatic assays were performed with increasing concentrations of P-EA , 200 μM SAM, 1 mM MnSO4, 0.5 μM BsAda, 4.72 μM SAHN and 2.5 μM PfPMT in a HEPES assay buffer, along with the inhibitor AQ . A reciprocal plot was generated from the absorbance readings at 265 nm versus the P-EA concentrations for each concentration of AQ. The fitted line was extrapolated through the X axis to determine the estimated Ki.All inhibitor containing stock solutions were prepared in Hpem1Δpem2Δ-pYes2.1-PfPMT yeast strains were pre-grown overnight in uracil dropout synthetic galactose (4%) (SG-ura) medium supplemented with 10 μM ethanolamine. The next day, cells were harvested by centrifugation, washed twice by resuspension in water and diluted to an A600 = 0.005 in fresh SG-ura medium supplemented with 100 μM ethanolamine, in the absence or presence of 10, 50, or 100 μM AQ. In a second set of experiments BY4741-pYes2.1, pem1Δpem2Δ-pYes2.1 and pem1Δpem2Δ-pYes2.1-PfPMT yeast strains were pre-grown overnight in SG-ura medium supplemented with 2 mM ethanolamine. The next day, cells were harvested by centrifugation, washed by resuspension in water and diluted to an A600 = 0.005 in fresh SG-ura medium lacking or supplemented with 1 mM choline in the presence of 0 or 200 μM AQ. In both sets of experiments, cells were grown at 30°C and monitored by measuring the A600.BY4741-pYes2.1 (wild-type) and mutant pem1Δpem2Δ-pYes2.1 and pem1Δpem2Δ-pYes2.1-PfPMT yeast strains were pre-grown in SG-ura medium supplemented with 2 mM ethanolamine and 2 mM choline for 24 hours. The cells were harvested by centrifugation, washed twice by resuspension in water and diluted to an A600 = 0.03 in the SG-ura medium supplemented with 2 mM ethanolamine and grown overnight to an A600 ~ 1.5. The cells were next harvested and the lipids extracted for two dimensional thin layer chromatography using previously described methods [ methods . Lipids  methods . The resE. coli cells expressing 6his-epitope-tagged PfPMT were grown in minimal medium (M9) containing 15 N-ammonium chloride. The purification of the recombinant enzyme followed the same procedure described above except for the addition of a second step of purification through a Superdex G75 gel filtration column in a buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM DTT pH 6.9. The sample was concentrated and D2O was added to a final concentration of 7% (v/v). Amodiaquine stock solution was prepared in ddH2O due to its low solubility. Serial dilutions were prepared and 5 μl of the diluted samples were added to the NMR sample containing 310 μM of purified PfPMT. The AQ concentration was determined spectrophotometrically using extinction coefficients ε238 = 27 × 103M-1cm-1, ε251 = 23 × 103M-1cm-1 and ε341 = 19 × 103M-1cm-1 [1H-15N HSQC experiments were performed on 15N-labeled PfPMT using a Varian Inova spectrometer operating at a 1H frequency of 600 MHz. Data were processed using nmrPipe [http://rnmrtk.uchc.edu.For NMR analysis, 3M-1cm-1 . The PfP3M-1cm-1 . Standar nmrPipe  and analTo identify new inhibitors of PfPMT we adapted an assay that couples the SAM-dependent transmethylation of P-EA by PfPMT to the activity of two enzymes SAH nucleosidase (SAHN) and adenine deaminase (BsAda) Fig. . PfPMT ain vitro and P. falciparum proliferation inside red blood cells with similar efficacy [in vitro and in vivo [To assess the applicability of the enzyme-coupled assay for screening inhibitors of PfPMT, the effects of some known or potential inhibitors of this enzyme were examined. As most drug screens are performed in the presence of DMSO, we first tested the effect of this agent on PfPMT activity. DMSO up to 10% had no effect on PfPMT activity (data not shown). The hexadecylphosphocholine analog, miltefosine (HePC), was previously reported to inhibit PfPMT activity efficacy . Using t in vivo -26 were P. falciparum within human erythrocytes at a concentration as high as 1 μM (data not shown). The specificity of inhibition of PfPMT by AQ was further demonstrated using its analog and antimalarial drug, chloroquine (CQ). At 200 μM concentration, CQ had no effect on PfPMT activity , a known inhibitor of the human phenylethanolamine-N-methyltransferase enzyme , which city Fig. . Other aity Fig. , suggestm PtdEtn ,30. In tm PtdEtn ,6. Examiium Fig. . AQ inhi0 h Fig. . These rded Fig. .pem1Δpem2Δ+PfPMT by AQ was due to the inhibition of the synthesis of PtdCho from ethanolamine, the synthesis of the major phospholipids PtdCho and PtdEtn of yeast membranes was examined in the absence or presence of AQ. Consistent with previous findings [pem1Δpem2Δ cells harboring an empty vector produced ~3% of total phospholipid as PtdCho after 5 to 6 generations of growth in the choline deficient medium, whereas those expressing PfPMT produced ~18% PtdCho . These enzymes catalyze the methylation of a large number of different substrates including DNA, RNAs, phospholipids, fatty acids, inositol, phosphoethanolamine, phenylethanolamine, tocopherol, catechol and histamine. Although some methyl donor molecules such as S-methylmethionine, betaine and folate can be used, the majority of biologically active methyltransferase enzymes utilize SAM as a methyl donor . SAM-metan et al  for the ed assay . In thisf 2.1 μM . Using tf 2.1 μM . Other mf 2.1 μM -39 had nfalciparum malaria cases in Africa suggested that AQ is more effective in clearing parasites, including CQ resistant strains, and resulted in better clinical recovery compared to CQ [-1 [In vitro both AQ and AQm inhibit parasite intraerythrocytic growth with IC50 values of 18.2 and 67.5 nM, respectively [PfCRT and PfMDR1 genes, whereas high AQ resistance involves unknown mechanisms [in vivo by using yeast as a surrogate system. In yeast, the growth of mutants that rely on PfPMT for survival was dramatically altered in the presence of this compound. Consistent with this phenotype, the synthesis of PtdCho in these complemented cells was strongly inhibited by AQ. Surprisingly, the amplitude of inhibition of pem1Δpem2Δ+PfPMT cells by AQ was dependent on the concentration of ethanolamine in the culture medium. This is likely due to the fact that these complemented cells grow faster on medium containing high concentrations of ethanolamine as a result of increased substrate availability for PfPMT.Amodiaquine is a structural analog of CQ. Analysis of uncomplicated ed to CQ -42. Amoded to CQ . Followio CQ [-1 . In the o CQ [-1 . Up to 9o CQ [-1 . In vitrectively . The modchanisms . These fP. falciparum intraerythrocytic development, multiplication and survival [A. thaliana PEAMT results in multiple morphological phenotypes, including pale-green leaves, early senescence, temperature-sensitive male sterility and increased sensitivity to salts [A. thaliana mutant with the T-DNA inserted at the At3g18000 locus (XIPOTL1), which encodes PEAMT revealed severe alterations in root developmental and induces cell death in root epidermal cells [C. elegans, studies aimed at silencing the PEAMT genes PMT-1 and PMT-2 demonstrated an essential function of these genes in worm, growth, development and survival [Recent genetic studies have shown that PfPMT plays an important function during survival , and an to salts . Furtheral cells . In C. esurvival . AltogetP. falciparum parasites lacking PfPMT were, however, equally sensitive to AQ as wild-type parasites in medium containing choline. This result suggests that PfPMT is not the sole target of AQ in choline-containing medium. Our finding that AQ inhibits PfPMT activity, and the fact that this compound also inhibits other eukaryotic methyltransferases, suggest that it may exert its antimalarial activity by targeting several parasite methyltransferases. Several studies have suggested that aminoquinolines may exert their antimalarial activity by interfering with biological functions other than heme detoxification in the digestive vacuole. Famin and Ginsburg reported inhibition of parasite's 6-phosphogluconate dehydrogenase by CQ [P. falciparum strains revealed that rings (prior to the formation of the digestive vacuole) and schizont stage parasites (after hemoglobin degradation and heme detoxification activities have plateaued) were equally sensitive to this compound [P. falciparum have not been investigated, our results indicate that at its peak plasma concentration, AQ is likely to inhibit PfPMT and possibly other parasite methyltransferases. Future studies will aim to identify Plasmodium methyltransferases inhibited by AQ and to characterize the mechanism of sensitivity and resistance to AQ. From a chemistry standpoint, AQ represents an excellent lead compound for the rational design of better inhibitors of PfPMT and other parasite methyltransferases as well as plant and nematode PMTs.se by CQ . Sharma se by CQ . Recentlse by CQ , and to se by CQ . More recompound . The bolcompound . TogetheHorton and coworkers have previously shown that AQ acts both as a competitive and a noncompetitive inhibitor of human HNMT . The cryThe enzyme-coupled assay adapted for PfPMT is a simple and reliable method for measuring PfPMT activity and screening for specific inhibitors of this enzyme. The identification of AQ as an inhibitor of PfPMT may help in the future design of compounds that specifically inhibit this enzyme and possibly other PMTs.AQ: amodiaquine AQm: desethylamodiaquine CQ: chloroquine PtdCho: phosphatidylcholine; PtdEtn: phosphatidylethanolamine; PtdSer: phosphatidylserine; P-EA: phosphoethanolamine; PMT: phosphoethanolamine methyltransferase; SDPM: serine decarboxylase phosphoethanolamine methyltransferase; CDP: citidylyldiphosphate; PfCCT: CDP-choline cytidylyl-transferase; PfCEPT: CDP-diacylgylcerol-choline phosphotransferase; SAM: s-adenosylmethionine; TLC: thin layer chromatography; SAH: s-adenosylhomocystein; HNMT: histamine methyltransferase; NMR: nuclear magnetic resonance; SAHN: SAH nucleosidase.The authors declare that they have no competing interests.AB optimized the enzyme coupled assay, performed the enzyme inhibitor studies, prepared all the figures and assisted in the writing and editing of the manuscript and the revisions. AM contributed to the initial development of the enzyme coupled assay and cloning of the BsADA used in the study. She also helped in the design of experiments and writing of the manuscript. JC and DV conducted the yeast growth assays and the yeast phospholipid analyses. IR, BH and JH contributed to the structural modeling and NMR analysis of PfPMT-amodiaquine interaction and helped in the writing and editing of the manuscript. CBM conceived, established the experimental framework of the study, analyzed the data and contributed to the writing and editing of the manuscript. All authors read and approved the final manuscript.Fig. S1. Overlay of the full 1H-15N HSQC spectra of PfPMT in the absence (red) or presence of 0.06 (orange), 0.12 (yellow), 0.25 (green), 0.5 (blue) and 1 mM (purple) of AQ.Click here for fileFig. S2. Sequence alignment of HNMT and PfPMT. Residues that are identical, conserved, and semi-conserved are indicated by asterisk, colon, and period, respectively. The AQ-interacting residues are colored as in Fig. Click here for file


Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis.Knockdown resistance (An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations (kdr-e and kdr-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population.A total of 52 pyrethroid and DDT resistant An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr-e and kdr-w resistance mutations, seven specimens (13.5%) were homozygous for the kdr-e mutation, and 14 specimens (26.9%) were homozygous for the kdr-w mutation. A single individual was genotyped as heterozygous kdr-e mutation (1.9%) only and another as heterozygous kdr-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr-w, six specimens (11.5%) homozygous for the kdr-e mutation, one specimen (1.9%) heterozygous for the kdr-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis.Fifty-one specimens were found to be kdr-e and kdr-w mutations were abundant in An. gambiae S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both kdr-e and kdr-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of kdr is still held as the gold standard assay, this paper highlights the importance of kdr mutation confirmation via sequencing especially in regions where kdr frequency has never been reported before or where both the kdr-e and kdr-w mutations are present simultaneously.Both the  One of the main strategies for malaria vector control is the use of pyrethroid insecticides for indoor residual spraying (IRS) and for the impregnation of long-lasting bed nets (LLINs). Reports of insecticide resistance in the major African malaria vectors have increased dramatically over the past few years -13. The Pyrethroids and organochlorines (eg. DDT) target the para-type sodium channel gene in the insect nervous system ,15. Crospara-type sodium channel gene have been reported in An. gambiae. Martinez-Torres et al [kdr mutation in An. gambiae from West Africa leads to the substitution of a leucine (TTA) for phenylalanine (TTT) . Ranson et al [kdr mutation in the same amino acid position in An. gambiae from Kenya. This mutation causes a substitution from leucine (TTA) to serine (TCA)  and is referred to as the East African mutation or kdr-e. More recently studies have reported heterozygous substitutions for both kdr mutations in An. gambiae from Cameroon  [kdr mutations, the hydrolysis probe analysis can be considered to be one of the best [kdr mutations should be screened concurrently. Changing default setting on machine may further enhance the accuracy of results.Currently, there are several different assays available for screening the DNA substitutions related to the (AS-PCR) ,22. Howe(AS-PCR) , Heated (AS-PCR) , Fluores(AS-PCR) , PCR elo(AS-PCR) , Sequenc(AS-PCR) , hydroly(AS-PCR) , Primer (AS-PCR) , multipl(AS-PCR)  and pyro(AS-PCR) . With rethe best . This stAn. gambiae s.l. using the keys of Gillies and Coetzee [et al [et al [et al [Fifty two mosquitoes were collected indoors in Pointe-Noire, Republic of the Congo (Congo-Brazzaville)  in April 2009 as part of a survey for insecticide resistance. The specimens were identified as  Coetzee . DNA wase [et al . DNA conl [et al  was usedl [et al .et al [kdr-Forward (5'-CATTTTTCTTGGCCACTGTAGTGAT-3') and kdr-Reverse (5'-CGATCTTGGTCCATGTTAATTTGCA-3') were used for binding the flanking region of both kdr mutation sites in the sodium channel gene. The probe WT (5'-CTTACGACTAAATTTC-3') was labelled with VIC for the detection of the wildtype allele and the probes kdrE (5'-ACGACTGAATTTC-3') and kdrW (5'-ACGACAAAATTTC-3') were labelled with 6-FAM for detection of the kdr-e and kdr-w resistant alleles respectively. The two standard primers (kdr-Forward and kdr-Reverse) and the WT probe were used either in one reaction with the kdrE probe for detecting kdr-e or in another reaction with the kdrW probe for detecting kdr-w. The 20 μL PCR reaction contained 1 μL of the genomic DNA of an individual mosquito, 10 μL of IQ™Supermix (Bio-Rad), 0.8 μM of each primer and 0.4 μM of each probe. The PCR cycling conditions were as follows: an initial denaturation at 95°C for 10 minute, followed by 40 cycles of 95°C for 10 seconds and 60°C for 45 seconds. The increase in VIC and FAM fluorescence was monitored in real time by detecting fluorescence of VIC (560-580 nm detection) and FAM (515-530 nm detection) channels for each dye respectively. The assay was repeated at least twice to ensure that experimental error was limited.The protocol of Bass et al  was usedkdr-e and kdr-w mutations region in the para-type sodium channel gene from 52 individuals was amplified using Agd1 (5'-ATAGATTCCCCGACCATG-3') and Agd2 (5'-AGACAAGGATGATGAACC-3') primers [2, 0.2 mM of each dNTP, 0.4 μM of each primer, and 1 unit of Taq DNA polymerase. The cycling conditions were as follows: a 2 minute 94 °C denaturation step; 40 cycles of 30 seconds at 94 °C, 30 seconds at 50°C and 30 seconds at 72°C; final extension for 5 minutes at 72°C. The PCR products were electrophoresed on 1.5% agarose gel containing 0.5 μg/mL ethidium bromide. Direct PCR sequencing was performed by MACROGEN  for both strands using the Adg1 and Agd2 primers. The results were aligned and analysed using Clustal X and Bioedit.Genotypes of individuals were confirmed by DNA sequencing. A segment of the  primers . A totalAn. gambiae S-forms and one individual was identified as a hybrid M/S. This was confirmed using DNA sequencing as well as the method of Fanello et al [kdr mutations. No M-form specimens were identified in this study. Comparison between the hydrolysis probe analysis  and DNA sequencing revealed that only 51.9% for the kdr-e and 86.5% for the kdr-w gave similar results  from the original default axes as determined by the hydrolysis probe analysis programme were performed  of specimens carried both the kdr-e and kdr-w mutations in heterozygote form. Seven specimens were homozygous for the kdr-e mutation and 14 homozygous for the kdr-w mutation. There was only one specimen heterozygous for the kdr-e mutation and one other that was heterozygous for the kdr-w mutation. Sequence analysis did not reveal any homozygous susceptible specimens for both kdr mutations i.e. the wild type form.The region for the An. gambiae S-form (98.1%). Unexpectedly, one specimen was identified as a hybrid M/S whilst no An. gambiae M-form were collected in this study area. Koekemoer et al (Multiple insecticide resistance in Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo, submitted) published additional data from this area from 343 specimens. This was the only hybrid identified from large sample analysis and additional genetic studies would need to be concluded to explain the presence of the hybrid in the rare presence of An. gambiae M-form (0.3%) by performing analysis of large sample size. The presence of the kdr mutations within this study cohort was unexpectedly high . Both kdr-e and kdr-w resistance mutations occurred simultaneously in 29 (55.8%) out of 52 specimens. The frequencies of the homozygous kdr-e and kdr-w single mutations were 7 (13.5%) and 14 specimens (26.9%) respectively. There was one specimen heterozygous for the kdr-e (1.9%) and another heterozygous for the kdr-w (1.9%) mutations. Although recent studies reported that the frequencies of the both mutations present at the same time were much lower , deltamethrin (26%), dieldrin (69%) and bendiocarb (3%)  from Pointe Noire, Republic of the Congo, submitted). This study also suggested that both kdr mutations in An. gambiae are widespread in the region.Most of the specimens collected in Pointe-Noire were  S-form 9.1%. Unexkdr mutations, it is much more time consuming and expensive (> US$10 per sample for sequencing in both directions) than other assays. Bass et al [kdr mutations (5.2% of failed reactions and 0% of mis-scores) when compared to AS-PCR, HRM, HOLA, SSOP-ELISA and PCR-Dot Blot assays even though the cost of the assay (US$1.72 per sample) is higher than some of the other methods (US$0.62 - US$ 1.74 per sample). In this study, 4% (2/52) samples gave different results between the hydrolysis probe and sequencing assays due to a failed reaction of either the kdr-e or kdr-w PCR.Although DNA sequencing remains the most accurate method for detecting the presence of ss et al  reportedResults from this study recommend that the allelic discrimination axes used for detecting both mutations simultaneously can be adjusted to increase sensitivity of the assay. This is especially true in those populations carrying both mutations at high frequency. However, this decision should be made cautiously and sequencing should be used to validate the adjustments made.kdr genotyping. The best method for genotyping seems to be end point analysis as well as intensity of fluorescence, although two out of fifty two samples in this study could not be genotyped using these values due to misleading RFU values, even after adjustment of the default allelic discrimination axes.The accuracy of the assay could fluctuate due to the determination of the allelic discrimination values for the axes as well as due to other variable running conditions. A number of conditions such as quality and quantity of DNA templates, master mixes, PCR assay performers, the positioning of the allelic discrimination axes and other conditions might affect kdr mutations in An. gambiae and the hydrolysis probe analysis is a preferred method. As this assay becomes more widely used in African laboratories it is important to emphasize that accurate genotyping in An. gambiae will rely on concurrent testing of both kdr-e and kdr-w mutations. In addition, this should be noted that default settings might have to be adjusted to enhance results.Various new assays have been developed to screen The authors declare that they have no competing interests.An. gambiae, kdr-e and kdr-w assays and DNA sequence, analysed the data and drafted the manuscript. BLS contributed to the design of this study, carried out identification of species, repetition of both kdr mutation assays, helped analyse the data and draft the manuscript. RHH collected all specimens from Pointe-Noire, identified species morphologically and helped draft the manuscript. LLK and MC assisted in data analysis and drafting of the manuscript.KSC carried out identification of species and molecular forms of All authors have read and approved the final manuscript.


Bisphenol A (BPA), a well-known endocrine disruptor, is highly glucuronidated in the liver, and the resultant BPA-glucuronide (BPA-GA) is excreted primarily into bile. However, in rodents, prenatal exposure to low doses of BPA can adversely affect the fetus, despite the efficient drug-metabolizing systems of the dams. The transport mechanisms of BPA from mother to fetus are unknown.To test our hypothesis that BPA-GA—an inactive metabolite—is passed through the placenta to the fetus, where it affects the fetus after reactivation, we investigated the placental transfer of BPA-GA and reactivation to BPA in the fetus.After performing uterine perfusion with BPA-GA in pregnant rats, we examined the expression and localization of the placental transporters for drug metabolites in the perfusate by reverse-transcriptase polymerase chain reaction and immunohistochemistry. We also investigated the deconjugation of BPA-GA in the fetus and examined uridine 5′-diphospho-glucuronosyltransferase (UGT) activity toward BPA and the expression of UGT isoforms in fetal liver.We detected BPA-GA and deconjugated BPA in the fetus and amniotic fluid after perfusion. In the trophoblast cells, organic anion-transporting polypeptide 4a1 (Oatp4a1) was localized on the apical membrane, and multidrug resistance-associated protein 1 (Mrp1) was localized to the basolateral membrane. We observed deconjugation of BPA-GA in the fetus; furthermore, we found the expression of UGT2B1, which metabolizes BPA, to be quite low in the fetus.These results demonstrate that BPA-GA is transferred into the fetus and deconjugated in the fetus because of its vulnerable drug-metabolizing system. Leaching of BPA out of the products is increased by heating , contactin vitro . Severalin vitro , nervousin vitro , and immin vitro .Generally, adult animals are able to metabolize and eliminate BPA from the body. Previously, we found that BPA is highly glucuronidated by UGT2B1, an isoform of uridine 5′-diphospho-glucuronosyltransferase (UGT) expressed in the rat liver . In addi14C-BPA, 14C-BPA  were purchased from Sankyo Lab Co. . Animals were housed individually under a 12/12-hr light/dark cycle and had  Animals .A schematic illustration of uterine perfusion is shown in 2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 26 mM NaHCO3, 2.5% dextran from Leuconostoc mesenteroides, and 3% Dextran 70) as the perfusate. The perfusate was pumped at a constant rate of 3 mL/min. First, we perfused four animals in a preliminary group with 10 μM BPA-GA for 20 min to determine whether BPA-GA is transferred into the fetus across the placenta. In this group, we performed a preliminary perfusion with mKRB for 5 min to wash away blood, followed by 20 min inflow of mKRB containing 10 μM BPA-GA. After the perfusion, the fetus and amniotic fluid were collected.We used modified Krebs-Ringer’s buffer . From the beginning of the BPA-GA perfusion, the perfusate that drained from the caudal vena cava was collected from each animal at 5-min intervals for 90 min. At the end of perfusate collection, the fetus, amnion, amniotic fluid, uterus, and placenta were collected. We also performed a control study using 1-naphthol-glucuronide  as the substrate. Four pregnant rats were used in each perfusion group. Values are expressed as mean ± SE.Perfusates collected from the caudal vena cava were collected after uterine perfusion and prepared for HPLC and LC/TOF-MS analysis. Each perfusate sample was mixed with a 4-fold volume of acetonitrile and centrifuged at 13,000 rpm for 5 min at 4°C; each supernatant was then analyzed by HPLC.To prepare tissues for HPLC and LC/TOF-MS analysis, we first added each sample to 0.3 mL methanol; the sample was homogenized and then sonicated for 5 min. The products were centrifuged at 13,000 rpm for 5 min at 4°C, and the supernatants were concentrated by solid-phase extraction using an Oasis HLB Plus cartridge . The extracts were then analyzed by HPLC.We used an HPLC system  that consisted of a dual pump (DP-8020), a fluorescent photometer (FS-8020), and a column oven (CO-8020). Samples were separated at 40°C using a reverse-phase column  at a flow rate of 1.0 mL/min under a linear gradient of solution A (methanol/water = 24/76 vol/vol with 10 mM ammonium acetate) and solution B (methanol) for 20 min. BPA-GA was detected at excitation/emission of 275/308 nm and 1-NA-GA was detected at excitation/emission of 283/336 nm. The results were recorded using LC-8020 integration software (Tosoh Corp.); the elution peaks of BPA and BPA-GA were noted and the concentrations compared with the standards.m/z 403–404 by TOF-MS. In the present study, the limits of detection of BPA and BPA-GA in LC/TOF-MS were both 20 pmol/mL (data not shown).LC/TOF-MS was performed using the HPLC system described above and LCT premier XE (Waters Corp.). The flow rate of HPLC was 0.3 mL/min, and BPA-GA was monitored at A23 anti-Mrp1 polyclonal antibody  was purchased from Enzo Life Sciences Inc. . We obtained anti–organic anion-transporting polypeptide (anti-Oatp4a1) polyclonal antibody from Scrum, Inc. ; anti-Oatp4a1 is designed to recognize the amino acid sequence LPSQSSA, which is common between human and rat .Placental tissue samples were analyzed by immunohistochemistry, as described in detail in the Supplemental Material (doi:10.1289/ehp.0901575). Briefly, tissue sections were incubated with anti-Oatp4a1 antibody or anti-Mrp1 antibody and analyzed with a confocal laser scanning microscope (Axionvert 200M) and PASCAL software .Maternal liver, placenta, fetal liver, and fetal intestine were collected on GD18.5. Total RNA was isolated from the tissues using the RNeasy Mini Kit  according to the manufacturer’s instructions. cDNA was synthesized from total RNA using Superscript III  reverse transcriptase according to the manufacturer’s instructions.Sequences of the oligonucleotide primers to amplify gene-specific cDNAs are available in the Supplemental Material (doi:10.1289/ehp.0901575).Mrp1, Mrp2, and Oatp4a1 mRNA in maternal liver and placenta was investigated by real-time RT-PCR using the QuantiTect SYBR Green PCR kit (Qiagen) and analyzed by iQ5/MyiQ Single-Color . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard. The copy number of each transporter gene was divided by that of GAPDH for normalization. Quantitative values are mean ± SE of four amplifications for placenta and three for maternal liver for each transporter gene.Quantitative expression of g at 4°C. After removing the supernatant, the pellets were resuspended in 5 mL William’s medium  containing collagenase  and DNase I  and incubated for 30 min at 37°C. After the incubation, samples were centrifuged for 5 min at 100 × g at 4°C, and the supernatants were removed. The remaining cells, which had been isolated from two fetuses, were resuspended in 200 μL William’s medium with or without 25 μM BPA-GA and incubated at 37°C for 10, 30, 60, or 120 min. At each time point, cells and medium were pooled and extracts were prepared for HPLC as described above, by adding the same volume of methanol. All experiments were repeated five times. Values express the mean ± SE.Fetal liver and heart tissues, collected on GD18.5, were washed twice with cold phosphate-buffered saline (PBS). Tissues for two fetuses were combined in one tube and then minced in cold PBS and centrifuged for 5 min at 100 × We examined UGT enzymatic activity using hepatic microsomes. We prepared the microsomes from rat liver and performed UGT enzyme analysis as described previously by First, we performed uterine perfusion  using BPNext, to examine the kinetics of BPA-GA in the maternal–placental–fetal unit, we performed perfusion for 20-min inflow with a lower concentration (2 μM) of BPA-GA and additional inflow without BPA-GA for 70 min , which deconjugates glucuronide conjugates, in both tissues , we did not detect 1-NA-GA or deconjugated 1-NA in the placenta or fetal tissues . These rOatp4a1 and Mrp1 in the placenta by quantitative RT-PCR. High expression levels of Oatp4a1 and Mrp1 in rat placenta have been also reported by Various transporters that either excrete drugs from the fetus to maternal blood or transfer nutrition and physiological compounds from the maternal body to the fetus have been isolated and characterized. Some of these transporters recognize xenobiotics because of their structural resemblance to physiological compounds. Generally, endocrine-disrupting chemicals, including BPA, often mimic endogenous chemical compounds such as estrogen. Moreover, the placenta produces steroid hormones that are used in both the mother and fetus . Therefo42− in the transport buffer, indicating that SO42− is not an inhibitor/substrate of the steroid sulfate transporters and that the carbon backbone of the steroid sulfates is a prerequisite for inhibition/transport. The selective transport between BPA-GA and 1-NA-GA, both glucuronide conjugates, could result from the difference in affinity not only for the conjugate residue but also for the parent residue. Further study is required to confirm the affinity of BPA-GA to these transporters. Placenta-specific expression of Oatp4a1 has already been reported . The possibility of transfer of placental deconjugated BPA could not be excluded completely in this experiment. Further work is required to examine the tissue-specific deconjugation of BPA-GA and the possibility of BPA transfer. At any rate, these data demonstrate that once BPA-GA is transferred into the fetus, it is deconjugated (reactivated) to BPA, and that this may be due to uptake of BPA-GA into fetal cells by transporters and subsequent catalysis by β-Gase.After co-incubation with BPA-GA, we detected deconjugated BPA in fetal liver and to a small degree in fetal heart . We obse tissues . InteresUGT2B1 mRNA in fetal organs was much lower than that in maternal liver. Moreover, in the UGT activity assay using liver microsomes on GD18.5, UGT activities in the fetal liver microsomes toward BPA were quite low. In this experiment, the ratio of fetal UGT activity toward BPA was about 20% of maternal microsomes . There are, however, some important differences between rodent and human placental structure. In rodents, maternal blood and fetal blood vessels are primarily separated by two syncytiotrophoblast layers, but in humans, only a syncytiotrophoblast monolayer separates fetal blood vessels from maternal blood . TherefoUGT2B15 was not expressed as well as other UGT isoforms examined. Both of these studies also suggest that human fetuses also have metabolizing systems vulnerable to BPA.In human liver microsomes, UGT2B15 is a major isoform that glucuronidates BPA . MoreoveUGT2B1 in the fetus. BPA may have been detected in the fetus after the perfusion due to high deconjugation ability of BPA-GA and low UGT activity toward BPA. Thus, we hypothesize that prenatal exposure to BPA may occur at levels sufficient to influence fetal development because of the exceptional drug-metabolizing system of the mother and fetus, which is particular to pregnancy. In 2003, 6 billion pounds of BPA were produced worldwide (Our study had three major findings. First, BPA-GA is transferred into the fetus through the placenta, even if only in small amounts. Second, BPA-GA is deconjugated to BPA in the fetus. Third, fetal liver microsomes have low ability to metabolize BPA to BPA-GA, as supported by the low expression of orldwide . Further